Nucleic acids encoding cytokine synthesis inhibitory factor (Interleukin-10)

ABSTRACT

Mammalian genes and proteins, designated cytokine synthesis inhibitory factors (CSIFs, now known as Interleukin-10 (IL-10)), are provided which are capable of inhibiting the synthesis of cytokines associated with delayed type hypersensitivity responses, and which, together with antagonists, are useful in treating diseases associated with cytokine imbalances, such as leishmaniasis and other parasitic infections, and certain immune disorders including MHC associated autoimmune diseases caused by excessive production of interferon-γ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/248,564,filed May 24, 1994, now abandoned, which is a continuation ofapplication Ser. No. 07/904,124, filed Jun. 25, 1992 and now abandoned,which is a divisional of application Ser. No. 07/546,235, filed Jun. 29,1990 and now abandoned.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions for treatingdiseases associated with immune system imbalances, particularlyimbalances involving humoral and cell-mediated immune responses. Theinvention also includes proteins and antagonists thereof capable ofmodulating the synthesis of certain cytokines involved in immune systemto achieve therapeutic effects.

BACKGROUND

Immune responses to antigen are classified as being predominantly eithercell-mediated, exemplified by the phenomena of delayed-typehypersensitivity (DTH), or humoral, exemplified by the production ofantibodies. Cell-mediated immunity is of paramount importance for therejection of tumors and for recovery from many viral, bacterial,protozoan, and fungal infections. In contrast, a humoral immune responseis the most effective form of immunity for eliminating toxins andinvading organisms from circulation. It has been observed that fordifferent antigens one or the other of these two responses oftenpredominates in a mutually exclusive fashion, and that the severity ofsome diseases, e.g. leprosy, leishmaniasis, and some types ofautoimmunity, may be due the inappropriate dominance of one class ofresponse over the other, Mosmann et al, Immunol. Today, Vol 8, pgs.223-227 (1987); Mosmann et al, Ann. Rev. Immunol., Vol. 7, pgs. 145-173(1989); Parish, Transplant. Rev, Vol. 13, pgs. 35-66 (1972); and Liew,Immunol. Today, Vol. 10, pgs. 40-45 (1989). It has further been observedthat sets of cytokines are separately associated with DTH reactions andhumoral immune responses, Cher et al, J. Immunol., Vol. 138, pgs.3688-3694 (1987); and Mosmann et al (1987 and 1989, cited above), and itis thought that diseases associated with these classes of response arecaused by the inappropriate production of the associated sets ofcytokines.

For example, a large body of evidence suggests that excessive productionof gamma interferon (IFN-γ) is responsible for major histocompatibilitycomplex (MHC) associated autoimmune diseases: Hooks et al, New EnglandJ. Med., Vol. 301, pgs. 5-8 (1979) (elevated serum levels of IFN-γcorrelated with autoimmunity); Basham et al, J. Immunol., Vol. 130, pgs.1492-1494 (1983) (IFN-γ can increase MHC gene product expression);Battazzo et al, Lancet, pgs. 1115-1119 (Nov. 12, 1983) (aberrant MHCgene product expression correlated with some forms of autoimmunity);Hooks et al, Ann. N.Y. Acad. Sci., Vol., pgs. 21-32 (1980) (higher IFN-γlevels correlated to greater severity of disease in SLE patients, andhistamine-release enhancing activity of interferon can be inhibited byanti-interferon sera); and Iwatani et al, J. Clin. Endocrin. andMetabol., Vol. 63, pgs. 695-708 (1986) (anti-IFN-γ monoclonal antibodyeliminated the ability of leucoagglutinin-stimulated T cells to induceHLA-DR expression). It is hypothesized that excess IFN-γ causes theinappropriate expression of MHC gene products which, in turn, causesautoimmune reactions against the tissues whose cells are inappropriatelyexpressing the MHC products and displaying autoantigens in the contextof the products.

In the area of clinical parasitology, it has recently been observed thatthe levels of IFN-γ and IL-2 are important factors in the progressionand/or resolution of the protozoan infection, leishmaniasis. Inparticular, the presence of adequate levels of IFN-γ appears to beessential for the activation of infected macrophages to eliminateintracellular amastigotes, Mauel and Behin, in Cohen et al, eds.,Immunology of Parasitic Infections (Blackwell, London, 1982). And, inmurine models of the disease, it has been shown that high levels ofIFN-γ and low levels of IL-4 are associated with resolution, whereas lowlevels of IFN-γ and high levels of IL-4 are associated with progressionof leishmaniasis, Heinzel et al, J. Exp. Med., Vol. 169, pgs. 59-72(1989).

In view of the above, it would be advantageous to have available agentsthat could shift the dominance of one class of immune response to theother, and in particular that could suppress or increase the synthesisof IFN-γ and/or other cytokines, respectively, as required for therapy.Such agents would be highly advantageous for treatment of diseasesassociated with inappropriate or inadequate immune responses, such astissue rejection, leishmaniasis and other parasitic diseases, and MHCassociated immune disorders including rheumatoid arthritis, systemiclupus erythematosus (SLE), myasthenia gravis, insulin-dependent diabetesmellitus, thyroiditis, and the like.

SUMMARY OF THE INVENTION

The present invention is directed to mammalian cytokine synthesisinhibitory factor (CSIF), CSIF analogs, CSIF peptides, and CSIFantagonists. It includes nucleic acids coding for polypeptidesexhibiting CSIF activity, as well as the polypeptides themselves, theiragonistic and/or antagonistic analogs, methods for their production, andmethods of using them to treat disorders associated with cytokineimbalances, particularly those leading to an inappropriate class ofimmune response. The invention also includes the use of CSIF or itsantagonists, alone or as vaccine adjuvants, to selectively induce apredominantly cell-mediated immune response or a predominantly humoralimmune response, respectively. Preferably, antagonists of CSIF arederived from monoclonal antibodies capable of blocking the biologicalactivity of CSIF. The nucleic acids of the invention are defined (1) bytheir homology to, or their ability to form detectable hybrids with, thecloned complementary DNA (cDNA) sequences disclosed herein, and (2) byfunctional assays for CSIF activity applied to the polypeptides encodedby the nucleic acids. As used herein, the term "CSIF activity" inreference to a protein or a polypeptide means that the protein orpolypeptide is capable of inhibiting or substantially reducing the levelof production of at least one of the following cytokines in the assaysdescribed below: IFN-γ, interleukin-2 (IL-2), lymphotoxin, interleukin-3(IL-3), or granulocyte-macrophage colony stimulating factor (GM-CSF).

A preferred embodiment of the invention is a mature human CSIF of theopen reading frame defined by the following amino acid sequence:

    MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

wherein the standard one-letter symbols for L-amino acids are listedleft to right starting from the N-terminal methionine. More preferably,the mature human CSIF is defined by the following amino acid sequence:

    SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

The invention is based in part on the discovery and cloning of cDNAswhich are capable of expressing proteins having CSIF activity.Accordingly, several such clones designated pcD(SRα)-F115 (carrying amouse CSIF gene), and pH5C and pH15C (each carrying a human CSIF gene)have been deposited with the American Type Culture Collection (ATCC),Rockville, Md. under the accession numbers 68027, 68191, and 68192,respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a dose-response relationship for the degree of IFN-γsynthesis inhibition in several mouse T cell clones treated withdifferent amounts of CSIF.

FIG. 2 is a diagram illustrating the major features of the mammalianexpression vectors pH5C and pH15C.

FIG. 3 illustrates the RBS-ATG-polylinker region of plasmidTAC-RBS-hCSIF.

FIGS. 4A-4C illustrate the nucleotide sequence of the cDNA insert ofpH15C.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes mature polypeptides, or proteins, of the largestopen reading frames of the cDNA inserts of pH5C, pH15C, pcD(SRα)-F115,and effectively homologous cDNAs, as well as antagonists thereof. Forsecreted proteins, an open reading frame usually encodes a polypeptidethat consists of a mature or secreted product covalently linked at itsN-terminus to a signal peptide. The signal peptide is cleaved prior tosecretion of the mature, or active, polypeptide. The cleavage site canbe predicted with a high degree of accuracy from empirical rules, e.g.von Heijne, Nucleic Acids Research, Vol. 14, pgs. 4683-4690 (1986), andthe precise amino acid composition of the signal peptide does not appearto be critical to its function, e.g. Randall et al, Science, Vol. 243,pgs. 1156-1159 (1989); Kaiser et al, Science, Vol. 235, pgs. 312-317(1987). Consequently, mature proteins are readily expressed by vectorsencoding signal peptides quite different than that encoded by the openreading frame defined by the cDNA inserts of pH5C, pH15C, andpcD(SRα)-F115.

I. Obtaining and Expressing CSIF cDNAs

The term "effectively homologous" as used herein means that thenucleotide sequence is capable of being detected by a hybridizationprobe derived from a cDNA clone of the invention. The exact numericalmeasure of homology necessary to detect nucleic acids coding for CSIFactivity depends on several factors including (1) the homology of theprobe to non-CSIF coding sequences associated with the target nucleicacids, (2) the stringency of the hybridization conditions, (3) whethersingle stranded or double stranded probes are employed, (4) whether RNAor DNA probes are employed, (5) the measures taken to reduce nonspecificbinding of the probe, (6) the nature of the method used to label theprobe, (7) the fraction of guanidine and cytosine bases in the probe,(8) the distribution of mismatches between probe and target, (9) thesize of the probe, and the like. Preferably, an effectively homologousnucleic acid sequence is at least ninety percent (90%) homologous to thecDNA of the invention. Most particularly, an effectively homologousnucleic acid sequence is one whose cDNA can be isolated by a probeconstructed from a cDNA insert of pcD(SRa)-F115, pH5C, pH15C, or anequivalent thereof, using the hybridization protocol described in theexamples with no more than a few false positive signals, e.g. less thana hundred. There is an extensive literature that provides guidance inselecting conditions for such hybridizations, e.g. Hames et al, NucleicAcid Hybridization: A Practical Approach (IRL Press, Washington, D.C.,1985); Gray et al, Proc. Natl. Acad. Sci., Vol. 80, pgs. 5842-5846(1983); Kafatos et al, Nucleic Acids Research, Vol. 7, pgs. 1541-1552(1979); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed.(Cold Spring Harbor Laboratory, New York, 1989); and Beltz et al, Meth.in Enzymol., Vol. 100, pgs. 266-285 (1983), to name a few.

Homology as the term is used herein is a measure of similarity betweentwo nucleotide (or amino acid) sequences. Homology is expressed as thefraction or percentage of matching bases (or amino acids) after twosequences (possibly of unequal length) have been aligned. The termalignment is used in the sense defined by Sankoff and Kruskal in chapterone of Time Warps, String Edits, and Macromolecules: The Theory andPractice of Sequence Comparison (Addison-Wesley, Reading, Mass., 1983).Roughly, two sequences are aligned by maximizing the number of matchingbases (or amino acids) between the two sequences with the insertion of aminimal number of "blank" or "null" bases into either sequence to bringabout the maximum overlap. Given two sequences, algorithms are availablefor computing their homology, e.g. Needleham and Wunsch, J. Mol. Biol.,Vol. 48, pgs. 443-453 (1970); and Sankoff and Kruskal (cited above) pgs.23-29. Also, commercial services and software packages are available forperforming such comparisons, e.g. Intelligenetics, Inc. (Palo Alto,Calif.); and University of Wisconsin Genetics Computer Group (Madison,Wis.).

Restriction endonuclease fragments of the vectors carrying the cDNAs ofthe invention are used to construct probes (using standard techniquessuch as nick-translation, e.g. see Sambrook et al., cited above) forscreening at low hybridization stringencies genomic or cDNA libraries(again, constructed by standard techniques) of a cell type suspected ofproducing CSIF. Standard screening procedures are employed, e.g.Grunstein et al., Proc. Natl. Acad. Sci., Vol. 72, pgs. 3961-3965(1975); or Benton et al., Science, Vol. 196, pgs. 180-183 (1977) or Woo,Methods in Enzymology, Vol. 68, pgs. 389-396 (1979). Alternatively,libraries can be screened with labeled oligonucleotide probes whosesequences are determined from the nucleotide sequences of the cDNAinserts of pcD(SRα)-F115, pH5C, and pH15C. Such probes can besynthesized on commercially available DNA synthesizers, e.g. AppliedBiosystems model 381A, using standard techniques, e.g. Gait,Oligonucleotide Synthesis: A Practical Approach, (IRL Press, WashingtonD.C., 1984). In either case, it is preferable that the probe be at least18-30 bases long. More preferably, the probe is at least 50-200 baseslong. Hybridization probes can also be used to screen candidate sourcesof CSIF mRNA prior to library construction.

A wide range of single-cell and multicellular expression systems (i.e.host-expression vector combinations) can be used to produce the proteinsof the invention. Possible types of host cells include, but are notlimited to, bacterial, yeast, insect, mammalian, and the like. Manyreviews are available which provide guidance for making choices and/ormodifications of specific expression systems, e.g. to name a few, deBoer and Shepard, "Strategies for Optimizing Foreign Gene Expression inEscherichia coli," pgs. 205-247, in Kroon, ed. Genes: Structure andExpression (John Wiley & Sons, New York, 1983), review several E. coliexpression systems; Kucherlapati et al., Critical Reviews inBiochemistry, Vol. 16, Issue 4, pgs. 349-379 (1984), and Banerji et al.,Genetic Engineering, Vol. 5, pgs. 19-31 (1983) review methods fortransfecting and transforming mammalian cells; Reznikoff and Gold, eds.,Maximizing Gene Expression (Butterworths, Boston, 1986) review selectedtopics in gene expression in E. coli, yeast, and mammalian cells; andThilly, Mammalian Cell Technology (Butterworths, Boston, 1986) reviewsmammalian expression systems. Likewise, many reviews are available whichdescribe techniques and conditions for linking and/or manipulatingspecific cDNAs and expression control sequences to create and/or modifyexpression vectors suitable for use with the present invention, e.g.Sambrook et al (cited above).

An E. coli expression system is disclosed by Riggs in U.S. Pat. No.4,431,739, which is incorporated by reference. A particularly usefulprokaryotic promoter for high expression in E. coli is the tac promoter,disclosed by de Boer in U.S. Pat. No. 4,551,433, which is incorporatedherein by reference. Secretion expression vectors are also available forE. coli hosts. Particularly useful are the pIN-III-ompA vectors,disclosed by Ghrayeb et al., in EMBO J., Vol. 3, pgs. 2437-2442 (1984),in which the cDNA to be transcribed is fused to the portion of the E.coli OmpA gene encoding the signal peptide of the ompA protein which, inturn, causes the mature protein to be secreted into the periplasmicspace of the bacteria. U.S. Pat. Nos. 4,336,336 and 4,338,397 alsodisclose secretion expression vectors for prokaryotes. Accordingly,these references are incorporated by reference.

Numerous stains of bacteria are suitable hosts for prokaryoticexpression vectors including strains of E. coli, such as W3110 (ATCC No.27325), JA221, C600, ED767, DH1, LE392, HB101, X1776 (ATCC No. 31244),X2282, RR1 (ATCC No. 31343) MRCI; strains of Bacillus subtilus; andother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcescens, and various species of Pseudomonas. General methods forderiving bacterial strains, such as E. coli K12 X1776, useful in theexpression of eukaryotic proteins is disclosed by Curtis III in U.S.Pat. No. 4,190,495. Accordingly this patent is incorporated byreference.

In addition to prokaryotic and eukaryotic microorganisms, expressionsystems comprising cells derived from multicellular organism may also beused to produce proteins of the invention. Of particular interest aremammalian expression systems because their posttranslational processingmachinery is more likely to produce biologically active mammalianproteins. Several DNA tumor viruses have been used as vectors formammalian hosts. Particularly important are the numerous vectors whichcomprise SV40 replication, transcription, and/or translation controlsequences coupled to bacterial replication control sequences, e.g. thepcD vectors developed by Okayama and Berg, disclosed in Mol. Cell.Biol., Vol. 2, pgs. 161-170 (1982) and Mol. Cell. Biol., Vol. 3, pgs.280-289 (1983), and improved by Takebe et al, Mol. Cell. Biol., Vol. 8,pgs. 466-472 (1988). Accordingly, these references are incorporatedherein by reference. Other SV40-based mammalian expression vectorsinclude those disclosed by Kaufman and Sharp, in Mol. Cell. Biol., Vol.2, pgs. 1304-1319 (1982), and Clark et al., in U.S. Pat. No. 4,675,285,both of which are incorporated herein by reference. Monkey cells areusually the preferred hosts for the above vectors. Such vectorscontaining the SV40 ori sequences and an intact A gene can replicateautonomously in monkey cells (to give higher copy numbers and/or morestable copy numbers than nonautonomously replicating plasmids).Moreover, vectors containing the SV40 ori sequences without an intact Agene can replicate autonomously to high copy numbers (but not stably) inCOS7 monkey cells, described by Gluzman, Cell, Vol. 23, pgs. 175-182(1981) and available from the ATCC (accession no. CRL 1651). The aboveSV40-based vectors are also capable of transforming other mammaliancells, such as mouse L cells, by integration into the host cell DNA.

Multicellular organisms can also serve as hosts for the production ofCSIF, e.g. insect larvae, Maeda et al, Nature, Vol. 315, pgs. 592-594(1985) and Ann. Rev. Entomol., pgs. 351-372 (1989); and transgenicanimals, Jaenisch, Science, Vol. 240, pgs. 1468-1474 (1988).

II. In Vitro Assays for CSIF

CSIF activity is the property of inhibiting the synthesis of at leastone cytokine in the group consisting of IFN-γ, lymphotoxin, IL-2, IL-3,and GM-CSF in a population of T helper cells induced to synthesize oneor more of these cytokines by exposure to syngeneic antigen presentingcells (APCs) and antigen. Preferably, the APCs are treated so that theyare incapable of replication, but that their antigen processingmachinery remains functional. This is conveniently accomplished byirradiating the APCs, e.g. with about 1500-3000 R (gamma or X-radiation)before mixing with the T cells.

Alternatively, cytokine inhibition may be assayed in primary or,preferably, secondary mixed lymphocyte reactions (MLR), in which casesyngeneic APCs need not be used. MLRs are well known in the art, e.g.Bradley, pgs. 162-166, in Mishell et al, eds. Selected Methods inCellular Immunology (Freeman, San Francisco, 1980); and Battisto et al,Meth. in Enzymol., Vol. 150, pgs. 83-91 (1987). Briefly, two populationsof allogenic lymphoid cells are mixed, one of the populations havingbeen treated prior to mixing to prevent proliferation, e.g. byirradiation. Preferably, the cell populations are prepared at aconcentration of about 2×10⁶ cells/ml in supplemented medium, e.g. RPMI1640 with 10% fetal calf serum. For both controls and test cultures, mix0.5 ml of each population for the assay. For a secondary MLR, the cellsremaining after 7 days in the primary MLR are re-stimulated by freshlyprepared, irradiated stimulator cells. The sample suspected ofcontaining CSIF may be added to the test cultures at the time of mixing,and both controls and test cultures may be assayed for cytokineproduction from 1 to 3 days after mixing.

Obtaining T cell populations and/or APC populations for CSIF assaysemploys techniques well known in the art which are fully described inDiSabato et al, eds., Meth. in Enzymol., Vol. 108 (1984). APCs for thepreferred CSIF assay are peripheral blood monocytes. These are obtainedusing standard techniques, e.g. as described by Boyum, Meth. inEnzymol., Vol. 108, pgs. 88-102 (1984); Mage, Meth. in Enzymol., Vol.108, pgs. 118-132 (1984); Litvin et al., Meth. in Enzymol., Vol. 108,pgs. 298-302 (1984); Stevenson, Meth. in Enzymol., Vol. 108, pgs.242-249 (1989); and Romain et al, Meth. in Enzymol., Vol. 108, pgs.148-153 (1984), which references are incorporated by reference.Preferably, helper T cells are used in the CSIF assays, which areobtained by first separating lymphocytes from the peripheral blood thenselecting, e.g. by panning or flow cytometry, helper cells using acommercially available anti-CD4 antibody, e.g. OKT4 described in U.S.Pat. No. 4,381,295 and available from Ortho Pharmaceutical Corp. Therequisite techniques are fully disclosed in Boyum, Scand. J. Clin. Lab.Invest., Vol. 21 (Suppl. 97), pg. 77 (1968); Meth. in Enzymol., Vol. 108(cited above), and in Bram et al, Meth. in Enzymol., Vol. 121, pgs.737-748 (1986). Generally, PBLs are obtained from fresh blood byFicoll-Hypaque density gradient centrifugation.

A variety of antigens can be employed in the assay, e.g. Keyhole limpethemocyanin (KLH), fowl γ-globulin, or the like. More preferably, inplace of antigen, helper T cells are stimulated with anti-CD3 monoclonalantibody, e.g. OKT3 disclosed in U.S. Pat. No. 4,361,549, in the assay.

Cytokine concentrations in control and test samples are measured bystandard biological and/or immunochemical assays. Construction ofimmunochemical assays for specific cytokines is well known in the artwhen the purified cytokine is available, e.g. Campbell, MonoclonalAntibody Technology (Elsevier, Amsterdam, 1984); Tijssen, Practice andTheory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and U.S. Pat.No. 4,486,530 are exemplary of the extensive literature on the subject.ELISA kits for human IL-2, human IL-3, and human GM-CSF are commerciallyavailable from Genzyme Corp. (Boston, Mass.); and an ELISA kit for humanIFN-γ is commercially available from Endogen, Inc. (Boston, Mass.).Polyclonal antibodies specific for human lymphotoxin are available fromGenzyme Corp. which can be used in a radioimmunoassay for humanlymphotoxin, e.g. Chard, An Introduction to Radioimmunoassay and RelatedTechniques (Elsevier, Amsterdam, 1982).

Biological assays of the cytokines listed above can also be used todetermine CSIF activity. A biological assay for human lymphotoxin isdisclosed in Aggarwal, Meth. in Enzymol., Vol. 116, pgs. 441-447 (1985),and Matthews et al, pgs. 221-225, in Clemens et al, eds., Lymphokinesand Interferons: A Practical Approach (IRL Press, Washington, D.C.,1987). Human IL-2 and GM-CSF can be assayed with factor dependent celllines CTLL-2 and KG-1, available from the ATCC under accession numbersTIB 214 and CCL 246, respectively. Human IL-3 can be assayed by itability to stimulate the formation of a wide range of hematopoietic cellcolonies in soft agar cultures, e.g. as described by Metcalf, TheHemopoietic Colony Stimulating Factors (Elsevier, Amsterdam, 1984).INF-γ can be quantified with anti-viral assays, e.g. Meager, pgs.129-147, in Clemens et al, eds. (cited above).

Cytokine production can also be determined by mRNA analysis.

Cytokine mRNAs can be measured by cytoplasmic dot hybridization asdescribed by White et al., J. Biol. Chem., Vol. 257, pgs. 8569-8572(1982) and Gillespie et al., U.S. Pat. No. 4,483,920. Accordingly, thesereferences are incorporated by reference. Other approaches include dotblotting using purified RNA, e.g. chapter 6, in Hames et al., eds.,Nucleic Acid Hybridization A Practical Approach (IRL Press, Washington,D.C., 1985). Generally, cytoplasmic dot hybridization involves anchoringmRNA from a cell or tissue sample onto a solid phase support, e.g.nitrocellulose, hybridizing a DNA probe to the anchored mRNA, andremoving probe sequences nonspecifically bound to the solid phasesupport or forming mismatched hybrids with the mRNA so that only probesequences forming substantially perfect hybrids with target mRNAsremain. The amount of DNA probe remaining is a measure of the number oftarget mRNA anchored to the solid phase support. The amount of DNA proberemaining is determined by the signal generated by its label.

Several standard techniques are available for labeling single and doublestranded nucleic acid fragments. They include incorporation ofradioactive labels, e.g. Harper et al., Chromosoma, Vol. 83, pgs.431-439 (1984); direct attachment of fluorescent labels, e.g. Smith etal., Nucleic Acids Research, Vol. 13, pgs. 2399-2412 (1985), andConnolly et al., Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985);and various chemical modifications of the nucleic acid fragments thatrender them detectable immunochemically or by other affinity reactions,e.g. Tchen et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3466-3470(1984); Richardson et al., Nucleic Acids Research, Vol. 11, pgs.6167-6184 (1983); Langer et al., Proc. Natl. Acad. Sci., Vol. 78, pgs.6633-6637 (1981); Brigati et al., Virology, Vol. 126, pgs. 32-50 (1983);Broker et al., Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); andBayer et al., Methods of Biochemical Analysis, Vol. 26, pgs. 1-45(1980).

Preferably mRNA from T cells is anchored for hybridization to the probeby the following protocol. Isolated T cells are lysed by suspending in alysis buffer (0.14M NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl pH 8.6, 0.5%Nonidet P-40 (a nonionic detergent, e.g. from Sigma)) at 4° C. at afinal concentration of 1×10⁸ cells/ml. The suspension is vortexed for 10sec and the nuclei are pelleted (13,000 g, 2.5 min). The resultingcytoplasmic lysates are then transferred to a sterile 1.5 ml tubecontaining 0.3 volumes of 20× SSC (1× SSC=0.15M NaCl, 0.015M trisodiumcitrate (standard saline citrate)) and 0.2 volumes of 37% (w/w)formaldehyde. The mixture is then incubated at 60° C. for 15 min andstored in aliquots at -70° C. For analysis, 15 ml of each sample istitered by serial three fold dilutions in 15× SSC into a 96-wellflat-bottomed microtiter plate (Falcon, Becton Dickinson, Oxnard,Calif.) in 0.1 ml. Each dilution is applied with suction to a sheet ofNytran (a modified nylon support available from Schleicher and Schuell,Keene, N.H.; 0.45 mm pore size) supported on a filter paper (Whatman 3mmChr, Whatman Inc., Clifton, N.J.) utilizing a 96 hold Minifoldapparatus (Schleicher and Schuell). The Nytran paper is then baked (80°C., 2 H) and treated with a prehybridization solution consisting of 50%formamide (BRL, Gaithersburg, Md.) 6× SSC, 50 mg/ml E. coli tRNA(Sigma), 0.2% (w/v) each of ficoll (MW=400,000), polyvinylpyrollidone,and bovine serum albumin (BSA). The probe is applied to the Nytransupport at a concentrate of about 50 ng probe/ml of prehybridizationsolution. Following hybridization, the support is washed two times for15 min each at room temperature in 2× SSC, then twice for 30 min each at60° C. in 2× SSC/0.5% SDS. The support is then exposed to film using anintensifying screen and quantitated by scanning with a laserdensitometer (e.g. Ultroscan XL, LKB Instruments Inc., Gaithersburg,Md.). If cytoplasmic dot hybridization lacks sufficient sensitivity,preferably the RNA is first extracted from the PBLs prior to blotting.For example, RNA may be extracted by the guanidinium thiocyanate methoddisclosed by Chirgwin et al., in Biochemistry, Vol. 18, pgs. 5294-5299(1979).

In some cases, samples to be tested for CSIF activity must be pretreatedto remove predetermined cytokines that might interfere with the assay.For example, IL-2 increases the production of IFN-γ in some cells. Thusdepending on the helper T cells used in the assay, IL-2 may have to beremoved from the sample being tested. Such removals are convenientlyaccomplished by passing the sample over a standard anti-cytokineaffinity column.

III. Monoclonal Antibodies and Antagonists Specific for CSIF

Preferably, antagonists of the invention are derived from antibodiesspecific for human CSIF. More preferably, the antagonists of theinvention comprise fragments or binding compositions specific for humanCSIF. Antibodies comprise an assembly of polypeptide chains linkedtogether by disulfide bridges. Two major polypeptide chains, referred toas the light chain and the heavy chain, make up all major structuralclasses (isotypes) of antibody. Both heavy chains and light chains arefurther divided into subregions referred to as variable regions andconstant regions. Heavy chains comprise a single variable region andthree different constant regions, and light chains comprise a singlevariable region (different from that of the heavy chain) and a singleconstant region (different from those of the heavy chain). The variableregions of the heavy chain and light chain are responsible for theantibody's binding specificity.

As used herein, the term "heavy chain variable region" means apolypeptide (1) which is from 110 to 125 amino acids in length, and (2)whose amino acid sequence corresponds to that of a heavy chain of amonoclonal antibody of the invention, starting from the heavy chain'sN-terminal amino acid. Likewise, the term "light chain variable region"means a polypeptide (1) which is from 95 to 115 amino acids in length,and (2) whose amino acid sequence corresponds to that of a light chainof a monoclonal antibody of the invention, starting from the lightchain's N-terminal amino acid.

As used herein the term "monoclonal antibody" refers to homogeneouspopulations of immunoglobulins which are capable of specifically bindingto human CSIF.

As used herein the term "binding composition" means a compositioncomprising two polypeptide chains (1) which, when operationallyassociated, assume a conformation having high binding affinity for humanCSIF, and (2) which are derived from a hybridoma producing monoclonalantibodies specific for human CSIF. The term "operationally associated"is meant to indicate that the two polypeptide chains can be positionedrelative to one another for binding by a variety of means, including byassociation in a native antibody fragment, such as Fab or Fv, or by wayof genetically engineered cysteine-containing peptide linkers at thecarboxyl termini. Normally, the two polypeptide chains correspond to thelight chain variable region and heavy chain variable region of amonoclonal antibody specific for human CSIF. Preferably, antagonists ofthe invention are derived from monoclonal antibodies specific for humanCSIF. Monoclonal antibodies capable of blocking, or neutralizing, CSIFare selected by their ability to inhibit CSIF-induced effects instandard CSIF bioassays, e.g. inhibition of IFN-γ synthesis.

Hybridomas of the invention are produced by well known techniques.Usually, the process involves the fusion of an immortalizing cell linewith a B-lymphocyte which produces the desired antibody. Alternatively,non-fusion techniques for generating an immortal antibody producing celllines are possible, and come within the purview of the presentinvention, e.g. virally induced transformation: Casali et al., "HumanMonoclonals from Antigen-Specific Selection of B Lymphocytes andTransformation by EBV," Science, Vol. 234, pgs. 476-479 (1986).Immortalizing cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine, and human origin. Mostfrequently, rat or mouse myeloma cell lines are employed as a matter ofconvenience and availability. Techniques for obtaining the appropriatelymphocytes from mammals injected with the target antigen are wellknown. Generally, either peripheral blood lymphocytes (PBLs) are used ifcells of human origin are desired, or spleen cells or lymph node cellsare used if non-human mammalian sources are desired. A host mammal isinjected with repeated dosages of the purified antigen, and the mammalis permitted to generate the desired antibody producing cells beforethese are harvested for fusion with the immortalizing cell line.Techniques for fusion are also well known in the art, and in general,involve mixing the cells with a fusing agent, such as polyethyleneglycol. Hybridomas are selected by standard procedures, such as HATselection. From among these hybridomas, those secreting the desiredantibody, i.e. specific for human CSIF, are selected by assaying theirculture medium by standard immunoassays, such as Western blotting,ELISA, RIA, CSIF neutralizing capability, or the like. Antibodies arerecovered from the medium using standard protein purificationtechniques, e.g. Tijssen, Practice and Theory of Enzyme Immunoassays(Elsevier, Amsterdam, 1985). Many references are available for guidancein applying any of the above techniques, e.g. Kohler et al., HybridomaTechniques (Cold Spring Harbor Laboratory, New York, 1980); Tijssen,Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985);Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984);Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications(CRC Press, Boca Raton, Fla., 1982); and the like. Hybridomas producingmonoclonal antibodies specific for human CSIF are then subjected to asecond screen using the CSIF assays described above to select onescapable of blocking, or neutralizing, the biological activity of CSIF.

The use and generation of fragments of antibodies is also well known,e.g. Fab fragments: Tijssen, Practice and Theory of Enzyme Immunoassays(Elsevier, Amsterdam, 1985); and Fv fragments: Hochman et al.Biochemistry, Vol. 12, pgs. 1130-1135 (1973), Sharon et al.,Biochemistry, Vol. 15, pgs. 1591-1594 (1976) and Ehrlich et al., U.S.Pat. No. 4,355,023; and antibody half molecules: Auditore- Hargreaves,U.S. Pat. No. 4,470,925.

Antibodies and antibody fragments characteristic of hybridomas of theinvention can also be produced by recombinant means by extractingmessenger RNA, constructing a cDNA library, and selecting clones whichencode segments of the antibody molecule, e.g. Wall et al., NucleicAcids Research, Vol. 5, pgs. 3113-3128 (1978); Zakut et al., NucleicAcids Research, Vol. 8, pgs. 3591-3601 (1980); Cabilly et al., Proc.Natl. Acad. Sci., Vol. 81, pgs. 3273-3277 (1984); Boss et al., NucleicAcids Research, Vol. 12, pgs. 3791-3806 (1984); Amster et al., NucleicAcids Research, Vol. 8, pgs. 2055-2065 (1980); Moore et al., U.S. Pat.No. 4,642,334; Skerra et al, Science, Vol. 240, pgs. 1038-1041 (1988);and Huse et al, Science, Vol. 246, pgs. 1275-1281 (1989). In particular,such techniques can be used to produce interspecific monoclonalantibodies, wherein the binding region of one species is combined withnon-binding region of the antibody of another species to reduceimmunogenicity, e.g. Liu et al., Proc. Natl. Acad. Sci., Vol. 84, pgs.3439-3443 (1987).

IV. Purification and Pharmaceutical Compositions

When polypeptides of the present invention are expressed in solubleform, for example as a secreted product of transformed yeast ormammalian cells, they can be purified according to standard proceduresof the art, including steps of ammonium sulfate precipitation, ionexchange chromatography, gel filtration, electrophoresis, affinitychromatography, and/or the like, e.g. "Enzyme Purification and RelatedTechniques," Methods in Enzymology, 22:233-577 (1977), and Scopes, R.,Protein Purification: Principles and Practice (Springer-Verlag, NewYork, 1982) provide guidance in such purifications. Likewise, whenpolypeptides of the invention are expressed in insoluble form, forexample as aggregates, inclusion bodies, or the like, they can bepurified by standard procedures in the art, including separating theinclusion bodies from disrupted host cells by centrifugation,solublizing the inclusion bodies with chaotropic and reducing agents,diluting the solubilized mixture, and lowering the concentration ofchaotropic agent and reducing agent so that the polypeptide takes on abiologically active conformation. The latter procedures are disclosed inthe following references, which are incorporated by reference: Winkleret al, Biochemistry, 25: 4041-4045 (1986); Winkler et al, Biotechnology,3: 992-998 (1985); Koths et al, U.S. Pat. No. 4,569,790; and Europeanpatent applications 86306917.5 and 86306353.3.

As used herein "effective amount" means an amount sufficient toameliorate a symptom of an autoimmune condition. The effective amountfor a particular patient may vary depending on such factors as the stateof the autoimmune condition being treated, the overall health of thepatient, method of administration, the severity of side-effects, and thelike. Generally, CSIF is administered as a pharmaceutical compositioncomprising an effective amount of CSIF and a pharmaceutical carrier. Apharmaceutical carrier can be any compatible, non-toxic substancesuitable for delivering the compositions of the invention to a patient.Generally, compositions useful for parenteral administration of suchdrugs are well known, e.g. Remington's Pharmaceutical Science, 15th Ed.(Mack Publishing Company, Easton, Pa. 1980). Alternatively, compositionsof the invention may be introduced into a patient's body by implantableor injectable drug delivery system, e.g. Urquhart et al., Ann. Rev.Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984); Lewis, ed. ControlledRelease of Pesticides and Pharmaceuticals (Plenum Press, New York,1981); U.S. Pat. No. 3,773,919; U.S. Pat. No. 3,270,960; and the like.

When administered parenterally, the CSIF is formulated in a unit dosageinjectable form (solution, suspension, emulsion) in association with apharmaceutical carrier. Examples of such carriers are normal saline,Ringer's solution, dextrose solution, and Hank's solution. Nonaqueouscarriers such as fixed oils and ethyl oleate may also be used. Apreferred carrier is 5% dextrose/saline. The carrier may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, e.g., buffers and preservatives. The CSIF ispreferably formulated in purified form substantially free of aggregatesand other proteins at a concentration in the range of about 5 to 20μg/ml. Preferably, CSIF is administered by continuous infusion so thatan amount in the range of about 50-800 μg is delivered per day (i.e.about 1-16 μg/kg/day). The daily infusion rate may be varied based onmonitoring of side effects and blood cell counts.

CSIF can be purified from culture supernatants of mammalian cellstransiently transfected or stably transformed by an expression vectorcarrying an CSIF gene. Preferably, CSIF is purified from culturesupernatants of COS 7 cells transiently transfected by the pcDexpression vector. Transfection of COS 7 cells with pcD proceeds asfollows: One day prior to transfection, approximately 10⁶ COS 7 monkeycells are seeded onto individual 100 mm plates in Dulbecco's modifiedEagle medium (DME) containing 10% fetal calf serum and 2 mM glutamine.To perform the transfection, the medium is aspirated from each plate andreplaced with 4 ml of DME containing 50 mM Tris.HCl pH 7.4, 400 mg/mlDEAE-Dextran and 50 μg of plasmid DNA. The plates are incubated for fourhours at 37° C., then the DNA-containing medium is removed, and theplates are washed twice with 5 ml of serum-free DME. DME is added backto the plates which are then incubated for an additional 3 hrs at 37° C.The plates are washed once with DME, after which DME containing 4% fetalcalf serum, 2 mM glutamine, penicillin (100 U/L) and streptomycin (100μg/L) at standard concentrations is added. The cells are then incubatedfor 72 hrs at 37° C., after which the growth medium is collected forpurification of CSIF. Alternatively, transfection can be accomplished byelectroporation as described in the examples. Plasmid DNA for thetransfections is obtained by growing pcD(SRα) containing the CSIF cDNAinsert in E. coli MC1061, described by Casadaban and Cohen, J. Mol.Biol., Vol. 138, pgs. 179-207 (1980), or like organism. The plasmid DNAis isolated from the cultures by standard techniques, e.g. Maniatis etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, New York, 1982).

When the antagonists of the inventions are derived from antibodies, theyare normally administered parenterally, preferably intravenously. Sincesuch protein or peptide antagonists may be immunogenic they arepreferably administered slowly, either by a conventional IVadministration set or from a subcutaneous depot, e.g. as taught byTomasi et al, U.S. Pat. No. 4,732,863. When administered parenterally,the antibodies and/or fragments are formulated in a unit dosageinjectable form in association with a pharmaceutical carrier, asdescribed above. The antibody is preferably formulated in purified formsubstantially free of aggregates, other proteins, endotoxins, and thelike, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml.

Selecting an administration regimen for an antagonist depends on severalfactors, including the serum turnover rate of the antagonist, the serumlevel of CSIF associated with the disorder being treated, theimmunogenicity of the antagonist, the accessibility of the target CSIF(e.g. if non-serum CSIF is to be blocked), the relative affinity of CSIFto its receptor(s) versus CSIF to the antagonist, and the like.Preferably, an administration regimen maximizes the amount of antagonistdelivered to the patient consistent with an acceptable level of sideeffects. Accordingly, the amount of antagonist delivered depends in parton the particular antagonist and the severity of the condition beingtreated. Guidance in selecting appropriate doses is found in theliterature on therapeutic uses of antibodies, e.g. Bach et al., chapter22, in Ferrone et al., eds., Handbook of Monoclonal Antibodies (NogesPublications, Park Ridge, N.J., 1985); and Russell, pgs. 303-357, andSmith et al., pgs. 365-389, in Haber et al., eds. Antibodies in HumanDiagnosis and Therapy (Raven Press, New York, 1977). Preferably,whenever the antagonist comprises monoclonal antibodies or Fab-sizedfragments thereof (including binding compositions), the dose is in therange of about 1-20 mg/kg per day. More preferably the dose is in therange of about 1-10 mg/kg per day.

V. Genetically Engineered Mutant CSIFs

Once nucleic acid sequence and/or amino acid sequence information isavailable for a native protein a variety of techniques become availablefor producing virtually any mutation in the native sequence, e.g.Shortle, in Science, Vol. 229, pgs. 1193-1201 (1985); Zoller and Smith,Methods in Enzymology, Vol. 100, pgs. 468-500 (1983); Mark et al., U.S.Pat. No. 4,518,584; Wells et al., in Gene, Vol. 34, pgs. 315-323 (1985);Estell et al., Science, Vol. 233, pgs. 659-663 (1986); Mullenbach et 20al., J. Biol. Chem., Vol. 261, pgs. 719-722 (1986), and Feretti et al.,Proc. Natl. Acad. Sci., Vol. 83, pgs.. 597-603 (1986). Accordingly,these references are incorporated by reference.

Muteins of the natural polypeptide may be desirable in a variety ofcircumstances. For example, undesirable side effects might be reduced bycertain muteins, particularly if the side effect activity is associatedwith a different part of the polypeptide from that of the desiredactivity. In some expression systems, the native polypeptide may besusceptible to degradation by proteases. In such cases, selectedsubstitutions and/or deletions of amino acids which change thesusceptible sequences can significantly enhance yields, e.g. Britishpatent application 2173-804-A where Arg at position 275 of human tissueplasminogen activator is replaced by Gly or Glu. Muteins may alsoincrease yields in purification procedures and/or increase shelf livesof proteins by eliminating amino acids susceptible to oxidation,acylation, alkylation, or other chemical modifications. For example,methionines readily undergo oxidation to form sulfoxides, which in manyproteins is associated with loss of biological activity, e.g. Brot andWeissbach, Arch. Biochem. Biophys., Vol. 223, pg. 271 (1983). Oftenmethionines can be replaced by more inert amino acids with little or noloss of biological activity, e.g. Australian patent applicationAU-A-52451/86. In bacterial expression systems, yields can sometimes beincreased by eliminating or replacing conformationally inessentialcystiene residues, e.g. Mark et al., U.S. Pat. No. 4,518,584.

Preferably cassette mutagenesis is employed to generate mutant proteins.A synthetic gene is constructed with a sequence of unique (when insertedin an appropriate vector) restriction endonuclease sites spacedapproximately uniformly along the gene. The unique restriction sitesallow segments of the gene to be conveniently excised and replaced withsynthetic oligonucleotides (i.e. "cassettes") which code for desiredmutations. Determination of the number and distribution of uniquerestriction sites entails the consideration of several factors including(1) preexisting restriction sites in the vector to be employed inexpression, (2) whether species or genera-specific codon usage isdesired, (3) the number of different non-vector-cutting restrictionendonucleases available (and their multiplicities within the syntheticgene), and (4) the convenience and reliability of synthesizing and/orsequencing the segments between the unique restriction sites.

The above technique is a convenient way to effect conservative aminoacid substitutions, and the like, in the native protein sequence."Conservative" as used herein means (i) that the alterations are asconformationally neutral as possible, that is, designed to produceminimal changes in the tertiary structure of the mutant polypeptides ascompared to the native protein, and (ii) that the alterations are asantigenically neutral as possible, that is, designed to produce minimalchanges in the antigenic determinants of the mutant polypeptides ascompared to the native protein. Conformational neutrality is desirablefor preserving biological activity, and antigenic neutrality isdesirable for avoiding the triggering of immunogenic responses inpatients or animals treated with the compounds of the invention. Whileit is difficult to select with absolute certainty which alternativeswill be conformationally and antigenically neutral, rules exist whichcan guide those skilled in the art to make alterations that have highprobabilities of being conformationally and antigenically neutral, e.g.Anfisen (cited above); Berzofsky, Science, Vol. 229, pgs. 932-940(1985); and Bowie et al, Science, Vol. 247, pgs. 1306-1310 (1990). Someof the more important rules include (1) substitution of hydrophobicresidues are less likely to produce changes in antigenicity because theyare likely to be located in the protein's interior, e.g. Berzofsky(cited above) and Bowie et al (cited above); (2) substitution ofphysiochemically similar, i.e. synonymous, residues are less likely toproduce conformational changes because the replacement amino acid canplay the same structural role as the substituted amino acid; and (3)alteration of evolutionarily conserved sequences is likely to producedeleterious conformational effects because evolutionary conservationsuggests sequences may be functionally important. In addition to suchbasic rules for selecting mutein sequences, assays are available toconfirm the biological activity and conformation of the engineeredmolecules. Biological assays for the polypeptides of the invention aredescribed more fully above. Changes in conformation can be tested by atleast two well known assays: the microcomplement fixation method, e.g.Wasserman et al., J. Immunol., Vol. 87, pgs. 290-295 (1961), or Levineet al. Methods in Enzymology, Vol. 11, pgs. 928-936 (1967) used widelyin evolutionary studies of the tertiary structures of proteins; andaffinities to sets of conformation-specific monoclonal antibodies, e.g.Lewis et al., Biochemistry, Vol. 22, pgs. 948-954 (1983).

VI. Human CSIF Peptide Antibodies

The invention includes peptides derived from human CSIF, and immunogenscomprising conjugates between carriers and peptides of the invention.The term immunogen as used herein refers to a substance which is capableof causing an immune response. The term carrier as used herein refers toany substance which when chemically conjugated to a peptide of theinvention permits a host organism immunized with the resulting conjugateto generate antibodies specific for the conjugated peptide. Carriersinclude red blood cells, bacteriophages, proteins, or syntheticparticles such as agarose beads. Preferably, carriers are proteins, suchas serum albumin, gamma-globulin, keyhole limpet hemocyanin,thyroglobulin, ovalbumin, fibrinogen, or the like.

Peptides of the invention are synthesized by standard techniques, e.g.Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed. (PierceChemical Company, Rockford, Ill., 1984). Preferably, a commercialpeptide synthesizer is used, e.g. Applied Biosystems, Inc. (Foster City,Calif.) model 430A. Peptides of the invention are assembled by solidphase synthesis on a cross-linked polystyrene support starting from thecarboxyl terminal residue and adding amino acids in a stepwise fashionuntil the entire peptide has been formed. The following references areguides to the chemistry employed during synthesis: Merrifield, J. Amer.Chem. Soc., Vol. 85, pg. 2149 (1963); Kent et al., pg 185, in Peptides1984, Ragnarsson, Ed. (Almquist and Weksell, Stockholm, 1984); Kent etal., pg. 217 in Peptide Chemistry 84, Izumiya, Ed. (Protein ResearchFoundation, B. H. Osaka, 1985); Merrifield, Science, Vol. 232, pgs.341-347 (1986); Kent, Ann. Rev. Biochem., Vol. 57, pgs. 957-989 (1988),and references cited in these latter two references.

In solid state synthesis it is most important to eliminate synthesisby-products, which are primarily termination, deletion, or modificationpeptides. Most side reactions can be eliminated or minimized by use ofclean, well characterized resins, clean amino acid derivatives, cleansolvents, and the selection of proper coupling and cleavage methods andreaction conditions, e.g. Barany and Merrifield, The Peptides, Cross andMeienhofer, Eds., Vol. 2, pgs 1-284 (Academic Press, New York, 1979). Itis important to monitor coupling reactions to determine that theyproceed to completion so that deletion peptides missing one or moreresidues will be avoided. The quantitative ninhydrin reaction is usefulfor that purpose, Sarin et al. Anal. Biochem, Vol. 117, pg 147 (1981).Na-t-butyloxycarbonyl (t-Boc)--amino acids are used with appropriateside chain protecting groups stable to the conditions of chain assemblybut labile to strong acids. After assembly of the protected peptidechain, the protecting groups are removed and the peptide anchoring bondis cleaved by the use of low then high concentrations of anhydroushydrogen fluoride in the presence of a thioester scavenger, Tam et al.,J. Amer. Chem. Soc., Vol. 105, pg. 6442 (1983). Side chain protectinggroups used are Asp(OBzl), Glu(OBzl), Ser(Bzl), Thr(Bzl), Lys(Cl-Z),Tyr(Br-Z), Arg(NGTos), Cys(4-MeBzl), and His(ImDNP). (Bzl, benzyl; Tostoluene sulfoxyl; DNP, dinitrophenyl; Im, imidazole; Z,benzyloxgycarbonyl). The remaining amino acids have no side chainprotecting groups. For each cycle the tBoc Na protected peptide-resin isexposed to 65 percent trifluoroacetic acid (from Eastman Kodak)(distilled before use) in dichloromethane (DCM), (Mallenckrodt): firstfor 1 minute then for 13 minutes to remove the Na-protecting group. Thepeptide-resin is washed in DCM, neutralized twice with 10 percentdiisopropylethylamine (DIEA) (Aldrich) in dimethylformamide (DMF)(Applied Biosystems), for 1 minute each. Neutralization is followed bywashing with DMF. Coupling is performed with the symmetric anhydride ofthe amino acid in DMF for 16 minutes. The symmetric anhydride isprepared on the synthesizer by dissolving 2 mmol of amino acid in 6 mlof DCM and adding 1 mmol of dicyclohexycarbodiimide (Aldrich) in 2 ml ofDCM. After 5 minutes, the activated amino acid is transferred to aseparate vessel and the DCM is evaporated by purging with a continuousstream of nitrogen gas. The DCM is replaced by DMF (6 ml total) atvarious stages during the purging. After the first coupling, thepeptide-resin is washed with DCM, 10 percent DIEA in DCM, and then withDCM. For recoupling, the same amino acid and the activating agent,dicyclohexylcarbodiimide, are transferred sequentially to the reactionvessel. After activation in situ and coupling for 10 minutes, sufficientDMF is added to make a 50 percent DMF-DCM mixture, and the coupling iscontinued for 15 minutes. Arginine is coupled as a hydroxybenzotriazole(Aldrich) ester in DMF for 60 minutes and then recoupled in the samemanner as the other amino acids. Asparagine and glutamine are coupledtwice as hydroxybenzotriazole esters in DMF, 40 minutes for eachcoupling. For all residues, the resin is washed after the secondcoupling and a sample is automatically taken for monitoring residualuncoupled α-amine by quantitative ninhydrin reaction, Sarin et al.(cited above).

The general technique of linking synthetic peptides to a carrier isdescribed in several references, e.g. Walter and Doolittle, "AntibodiesAgainst Synthetic Peptides," in Setlow et al., eds., GeneticEngineering, Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et al.Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al., Proc. Natl. Acad.Sci., Vol. 78, pgs. 3403-3407 (1981); Shimizu et al., U.S. Pat. No.4,474,754; and Ganfield et al., U.S. Pat. No. 4,311,639. Accordingly,these references are incorporated by reference. Also, techniquesemployed to link haptens to carriers are essentially the same as theabove-referenced techniques, e.g. chapter 20 in Tijsseu Practice andTheory of Enzyme Immunoassays (Elsevier, New York, 1985). The four mostcommonly used schemes for attaching a peptide to a carrier are (1)glutaraldehyde for amino coupling, e.g. as disclosed by Kagan and Glick,in Jaffe and Behrman, eds. Methods of Hormone Radioimmunoassay, pgs.328-329 (Academic Press, N.Y., 1979), and Walter et al. Proc. Natl.Acad. Sci., Vol. 77, pgs. 5197-5200 (1980); (2) water-solublecarbodiimides for carboxyl to amino coupling, e.g. as disclosed by Hoareet al., J. Biol. Chem., Vol. 242, pgs. 2447-2453 (1967); (3)bis-diazobenzidine (DBD) for tyrosine to tyrosine sidechain coupling,e.g. as disclosed by Bassiri et al., pgs. 46-47, in Jaffe and Behrman,eds. (cited above), and Walter et al. (cited above); and (4)maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for coupling cysteine(or other sulfhydryls) to amino groups, e.g. as disclosed by Kitagawa etal., J. Biochem. (Tokyo), Vol. 79, pgs. 233-239 (1976), and Lemer et al.(cited above). A general rule for selecting an appropriate method forcoupling a given peptide to a protein carrier can be stated as follows:the group involved in attachment should occur only once in the sequence,preferably at the appropriate end of the segment. For example, BDBshould not be used if a tyrosine residue occurs in the main part of asequence chosen for its potentially antigenic character. Similarly,centrally located lysines rule out the glutaraldehyde method, and theoccurrences of aspartic and glutamic acids frequently exclude thecarbodiimide approach. On the other hand, suitable residues can bepositioned at either end of chosen sequence segment as attachment sites,whether or not they occur in the "native" protein sequence. Internalsegments, unlike the amino and carboxy termini, will differsignificantly at the "unattached end" from the same sequence as it isfound in the native protein where the polypeptide backbone iscontinuous. The problem can be remedied, to a degree, by acetylating theα-amino group and then attaching the peptide by way of its carboxyterminus. The coupling efficiency to the carrier protein is convenientlymeasured by using a radioactively labeled peptide, prepared either byusing a radioactive amino acid for one step of the synthesis or bylabeling the completed peptide by the iodination of a tyrosine residue.The presence of tyrosine in the peptide also allows one to set up asensitive radioimmune assay, if desirable. Therefore, tyrosine can beintroduced as a terminal residue if it is not part of the peptidesequence defined by the native polypeptide.

Preferred carriers are proteins, and preferred protein carriers includebovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpethemocyanin (KLH), or the like. Peptides can be linked to KLH throughcysteines by MBS as disclosed by Liu et al., Biochemistry, Vol. 18, pgs.690-697 (1979). The peptides are dissolved in phosphate-buffered saline(pH 7.5), 0.1M sodium borate buffer (pH 9.0) or 1.0M sodium acetatebuffer (pH 4.0). The pH for the dissolution of the peptide is chosen tooptimize peptide solubility. The content of free cysteine for solublepeptides is determined by Ellman's method, Ellman, Arch. Biochem.Biophys., Vol. 82, pg. 7077 (1959). For each peptide, 4 mg KLH in 0.25ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg MBS(dissolved in dimethyl formamide) and stirred for 30 min at roomtemperature. The MBS is added dropwise to ensure that the localconcentration of formamide is not too high, as KLH is insoluble in >30%formamide. The reaction product, KLH-MBS, is then passed throughSephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0)to remove free MBS, KLH recovery from peak fractions of the columneluate (monitored by OD280) is estimated to be approximately 80%.KLH-MBS is then reacted with 5 mg peptide dissolved 25 in 1 ml of thechosen buffer. The pH is adjusted to 7-7.5 and the reaction is stirredfor 3 hr at room temperature. Coupling efficiency is monitored withradioactive peptide by dialysis of a sample of the conjugate againstphosphate-buffered saline, and ranged from 8% to 60%. Once thepeptide-carrier conjugate is available polyclonal or monoclonalantibodies are produced by standard techniques, e.g. as disclosed byCampbell, Monoclonal Antibody Technology (Elsevier, New York, 1984);Hurrell, ed. Monoclonal Hybridoma Antibodies: Techniques andApplications (CRC Press, Boca Raton, Fla., 1982); Schreier et al.Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980);U.S. Pat. No. 4,562,003; or the like. In particular, U.S. Pat. No.4,562,003 is incorporated by reference.

Both polyclonal and monoclonal antibodies can be screened by ELISA. Asin other solid phase immunoassays, the test is based on the tendency ofmacromolecules to adsorb nonspecifically to plastic. The irreversibilityof this reaction, without loss of immunological activity, allows theformation of antigen-antibody complexes with a simple separation of suchcomplexes from unbound material. To titrate antipeptide serum, peptideconjugated to a carrier different from that used in immunization isadsorbed to the wells of a 96-well microtiter plate. The adsorbedantigen is then allowed to react in the wells with dilutions ofanti-peptide serum. Unbound antibody is washed away, and the remainingantigen-antibody complexes are allowed to react with antibody specificfor the IgG of the immunized animal. This second antibody is conjugatedto an enzyme such as alkaline phosphatase. A visible colored reactionproduct produced when the enzyme substrate is added indicates whichwells have bound antipeptide antibodies. The use of spectrophotometerreadings allows better quantification of the amount of peptide-specificantibody bound. High-titer antisera yield a linear titration curvebetween 10⁻³ and 10⁻⁵ dilutions.

EXAMPLES

The following examples serve to illustrate the present invention.Selection of vectors and hosts as well as the concentration of reagents,temperatures, and the values of other variable parameters are only toexemplify application of the present invention and are not to beconsidered as limitations thereof.

Example I. Biological Activities of Mouse CSIF

Mouse CSIF-containing supernatants from the several T cell clones wereobtained by incubating the T cell clones (5×10⁶ cells/ml) in serum freemedium (RPMI 1640 lacking phenol red and containing 0.05 mM2-mercaptoethanol and 20 mM HEPES) and concanavalin A (5 μg/ml) for 24hours. The clones included cell lines, D9 described in U.S. Pat. No.4,613,459, D10 (described below), MB2-1 described in Mosmann et al, J.Immunol., Vol. 136, pgs. 2348-2357 (1986), CDC25 and CDC35 described inTony et al, J. Exp. Med., Vol. 161, pgs. 223- (1985), and M411-2 andM411-6. The T cell supernatants were assayed for their ability tosuppress IFN-γ synthesis in the cell line HDK-1, described inCherwinski, et al, J. Exp. Med., Vol. 166, pgs. 1229-1244(1987). Serialtwofold dilutions of samples from each T cell clone were prepared in96-wll flat-bottomed microtiter trays in a volume of 0.05 ml. HDK-1cells (5×10⁴ cells per well) along with irradiated (2500 R) syngeneicAPCs (spleen cells at 5×10⁵ cells per well) and antigen (keyhole limpethemocyanin at 150 μg/ml) were added in a volume of 0.15 ml. 11B11anti-IL-4 antibody (10 μg/ml), described in Ohara et, Nature, Vol. 315,pgs. 333-336 (1985), was added to samples suspected of containing IL-4.After incubation at 37° C. for 24 h, supernatants were collected andkept at 4° C. for periods of less than a week, or at -80° C. for longerperiods. Levels of IFN-γ were assayed by two site sandwich ELISA using arat anti-mouse IFN-γ monoclonal antibody, XMG1.2, and affinity-purifiedrabbit anti-mouse IFN-γ antibody. FIG. 1 shows the degree of inhibitionof IFN-γ synthesis as percentage of control levels.

CSIF produced by D10 cells was partially purified and applied to twodifferent T cell clones to examine the degree of cytokine synthesisinhibition as a function of CSIF concentration. The partially purifiedCSIF was prepared as follows: 1-2.5 L batches of concanavalin A-inducedD10 supernatant were concentrated approximately 10-fold using AmiconYM-5 membranes (Amicon Corp., Danvers, Mass.), passed through a 5-mlmannose-conjugated agarose column (E-Y Laboratories, San Mateo, Calif.),then further concentrated another 3- to 5-fold, for a totalconcentration of 30-50 fold. This material was then further purified bytwo steps of high performance liquid chromatography: first over ahydroxylapatite-based column (Bio-Gel HPHT, Bio-Rad Laboratories,Richmond, Calif.) and then over a gel filtration column (TSK-G 3000 SW,60 cm length, LKB Instruments, Gaithersburg, Md.). One such batch ofpartially purified CSIF was kept in aliquots at -80° C. and used as astandard of CSIF activity. When initially assayed, this preparationcaused approximately 50% inhibition of IFN-γ production at a dilution of1/200 in an assay volume of 0.2 ml, and so a standard unit was definedby assigning a value of 1000 U/ml to the standard CSIF preparation. Ineach assay below, the CSIF activity in unknown samples was quantitatedby comparing levels of inhibition of IFN-γ synthesis by the unknown tothat of the standard. The T cell clones that were assayed for inhibitionof cytokine synthesis were HDK-1 (described above) and MD13-10,described in Cell. Immunol., Vol. 97, pgs. 357- (1986). For the assay ofIL-3 and GM-CSF levels the partially purified CSIF was further treatedby passing it over anti-IL-3 and anti-GM-CSF affinity columns.Antibodies in 0.1 M NaCl, 0.1 M HEPES, and 0.08 M CaCl₂ were coupled toAffi-Gel 10 (Bio-Rad) at 4° C. with gentle mixing for 4 hours. Each 1-2ml column contained approximately 10 to 20 mg of coupled antibody.

As shown in the table below, IFN-γ production was inhibited in bothclones. The synthesis of the other cytokines, IL-2, lymphotoxin, IL-3,and GM-CSF was inhibited to a lesser degree or not at all in MD13-10cells.

                  TABLE                                                           ______________________________________                                                    % of Control Synthesis Level                                      Cell line                                                                             Cytokine  14 U/ml    42 U/ml                                                                              125 U/ml                                  ______________________________________                                        HDK-1   IFN-g     47.6       29.1   18.6                                         IL-2 71.7 59.6 40.4                                                           lymphotoxin 41.9 45.1 42.8                                                    IL-3 63.9 52.6 38.4                                                           GM-CSF 86.9 79.1 66.8                                                        MD13-10 IFN-g 36.0 27.5 23.2                                                   IL-2 88.2 109.3 96.0                                                          IL-3 60.2 63.0 51.0                                                           GM-CSF 109.0 119.9 97.6                                                    ______________________________________                                    

Example II. Construction of cDNA Library from D10 Cells and Isolation ofClone pcD(SRa)-F115

A cDNA library was constructed in the pcD(SRα) vector from mRNAextracted from D10 cells, described in Kaye et al, J. Exp. Med., Vol.158, pgs. 836- (1983), in accordance with the method of Okayama andBerg, Mol. Cell. Biol. 2: 161-170 (1982) and 3: 280-289 (1983), alsodisclosed in U.S. Pat. No. 4,695,542, which is incorporated byreference. The pcD(SRα) vectors carrying cDNA inserts were amplified inE. coli. Plasmid DNA was extracted from pools of these randomly pickedclones and used to transfect COS 7 monkey cells as described below. Thesupernatants of the COS 7 cultures were then tested for CSIF activity.COS cells were transfected as follows: One day prior to transfection,approximately 1.5×10⁶ COS 7 monkey cells were seeded onto individual 100mm plates in Dulbecco's modified Eagle medium (DME) containing 5% fetalcalf serum (FCS) and 2 mM glutamine. To perform the transfection, COS 7cells were removed from the dishes by incubation with trypsin, washedtwice in serum-free DME, and suspended to 10⁷ cells/ml in serum-freeDME. A 0.75 ml aliquot was mixed with 20 μg DNA and transferred to asterile 0.4 cm electroporation cuvette. After 10 minutes, the cells werepulsed at 200 volts, 960 μF in a BioRad Gene Pulser unit. After another10 minutes, the cells were removed from the cuvette and added to 20 mlof DME containing 5% FCS, 2 mM glutamine, penicillin, streptomycin, andgentamycin. The mixture was aliquoted to four 100 mm tissue culturedishes. After 12-24 hours at 37° C., 5% CO₂, the medium was replacedwith similar medium containing only 1% FCS and the incubation continuedfor an additional 72 hours at 37° C., 5% CO₂, after which the medium wascollected and assayed for CSIF activity. Subsequently, the sequence ofthe largest open reading frame of the cDNA insert of pcD(SRα)-F115 wasdetermined as follows:

    ATGCCTGGCT CAGCACTGCT ATGCTGCCTG CTCTTACTGA CTGGCATGAG                           - GATCAGCAGG GGCCAGTACA GCCGGGAAGA CAATAACTGC ACCCACTTCC                      - CAGTCGGCCA GAGCCACATG CTCCTAGAGC TGCGGACTGC CTTCAGCCAG                      - GTGAAGACTT TCTTTCAAAC AAAGGACCAG CTGGACAACA TACTGCTAAC                      - CGACTCCTTA ATGCAGGACT TTAAGGGTTA CTTGGGTTGC CAAGCCTTAT                      - CGGAAATGAT CCAGTTTTAC CTGGTAGAAG TGATGCCCCA GGCAGAGAAG                      - CATGGCCCAG AAATCAAGGA GCATTTGAAT TCCCTGGGTG AGAAGCTGAA                      - GACCCTCAGG ATGCGGCTGA GGCGCTGTCA TCGATTTCTC CCCTGTGAAA                      - ATAAGAGCAA GGCAGTGGAG CAGGTGAAGA GTGATTTTAA TAAGCTCCAA                      - GACCAAGGTG TCTACAAGGC CATGAATGAA TTTGACATCT TCATCAACTG                      - CATAGAAGCA TACATGATGA TCAAAATGAA AAGCTAA                             

and the amino acid sequence of the mature mouse CSIF protein determinedby the Heijne algorthm is as follows:

    QYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTD SLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGE KLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAM NEFDIFINCIEAYMMIKMKS

Example III. Screening cDNA Libraries for Human CSIF Using ProbesDerived from pcD(SRα)-F115: Isolation of pH5C and pH15C

A cDNA library constructed in pcD(SRα) from mRNA extracted from a humanT cell clone was screened with a collection of 70-mer oligonucleotideswhose sequences were complementary to the coding and noncoding strandsof the fragment of the mouse CSIF gene encoding mature CSIF. Standardhybridization protocols were used, e.g. bacterial colonies grown on 150mm petri dishes were transferred to GeneScreen membranes, treated withthe radioactively labeled oligonucleotide probes, washed, then exposedto X-ray film. The probes were hybridized under low stringencyconditions for the length of the probes: prehybridization consisted ofincubation of the target nucleic acids in 5× SET (20× SET is 3 MNaCl+0.4 Tris-Cl (pH 7.8)+20 mM EDTA) at 60° C., followed byhybridization under the same conditions, and washing in 5× SET at 50° C.Two clones carrying plasmids pH5C and pH15C were identified. Bothplasmids expressed proteins in COS 7 cells that were capable ofinhibiting IFN-γ synthesis in PHA-stimulated human PBLs. The cDNA insertof pH15C is illustrated in FIG. 4, and the nucleotide sequence of itslargest open reading frame is given below:

    5'- ATGCACAGCT CAGCACTGCT CTGTTGCCTG GTCCTCCTGA CTGGGGTGAG-                      -     GGCCAGCCCA GGCCAGGGCA CCCAGTCTGA GAACAGCTGC ACCCACTTCC-                 -     CAGGCAACCT GCCTAACATG CTTCGAGATC TCCGAGATGC CTTCAGCAGA-                 -     GTGAAGACTT TCTTTCAAAT GAAGGATCAG CTGGACAACT TGTTGTTAAA-                 -     GGAGTCCTTG CTGGAGGACT TTAAGGGTTA CCTGGGTTGC CAAGCCTTGT-                 -     CTGAGATGAT CCAGTTTTAC CTGGAGGAGG TGATGCCCCA AGCTGAGAAC-                 -     CAAGACCCAG ACATCAAGGC GCATGTGAAC TCCCTGGGGG AGAACCTGAA-                 -     GACCCTCAGG CTGAGGCTAC GGCGCTGTCA TCGATTTCTT CCCTGTGAAA-                 -     ACAAGAGCAA GGCCGTGGAG CAGGTGAAGA ATGCCTTTAA TAAGCTCCAA-                 -     GAGAAAGGCA TCTACAAAGC CATGAGTGAG TTTGACATCT TCATCAACTA-                 -     CATAGAAGCC TACATGACAA TGAAGATACG AAACTGA-3'                      

Example IV. Monoclonal Antibodies Specific for CSIF

A male Lewis rat is immunized with semi-purified preparations of COS7-cell expressed human CSIF. The rat is first immunized withapproximately 50 μg of human CSIF in Freund's Complete Adjuvant, andboosted twice with the same amount of material in Freund's IncompleteAdjuvant. Test bleeds are taken. The animal is given a final boost of 25μg in phosphate-buffered saline, and four days later the spleen isobtained for fusion.

Approximately 3×10⁸ rat splenocytes are fused with an equal number ofP3X63-AG8.653 mouse myeloma cells (available from the ATCC underaccession number CRL 1580). 3840 microtiter plate wells are seeded at5.7×10⁴ parental myeloma cells per well. Standard protocols for thefusion and subsequent culturing of hybrids are followed, e.g. asdescribed by Chretien et al, J. Immunol. Meth., Vol. 117, pgs. 67-81(1989). 12 days after fusion supernatants are harvested and screened byindirect ELISA on PVC plates coated with COS 7-produced human CSIF.Hybridoma JES3-19F1.1.1 was identified in this manner and deposited withthe American Type Culture Collection under accession number HB10487.

Hybridomas producing blocking antibodies are selected from the initiallyscreened hybridomas by their ability to produce antibodies thatcounteract the CSIF-induced inhibition of IFN-γ synthesis inPHA-stimulated human PBLs.

Example V. Expression of Human CSIF in a Bacterial Host

A synthetic human CSIF gene is assembled from a plurality of chemicallysynthesized double stranded DNA fragments to form an expression vectordesignated TAC-RBS-hCSIF. Cloning and expression are carried out in astandard bacterial system, for example E. coli K-12 strain JM101, JM103,or the like, described by Viera and Messing, in Gene, Vol. 19, pgs.259-268 (1982). Restriction endonuclease digestions and ligase reactionsare performed using standard protocols, e.g. Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York,1982).

The alkaline method (Maniatis et al., cited above) is used for smallscale plasmid preparations. For large scale preparations a modificationof the alkaline method is used in which an equal volume of isopropanolis used to precipitate nucleic acids from the cleared lysate.Precipitation with cold 2.5M ammonium acetate is used to remove RNAprior to cesium chloride equilibrium density centrifugation anddetection with ethidium bromide.

For filter hybridizations Whatman 540 filter circles are used to liftcolonies which are then lysed and fixed by successive treatments with0.5M NaOH, 1.5M NaCl; IM Tris.HCl pH8.0, 1.5M NaCl (2 min each); andheating at 80° C. 25 (30 min). Hybridizations are in 6×SSPE, 20%formamide, 0.1% sodium dodecylsulphate (SDS), 100 mg/ml E. coli tRNA,100 mg/ml Coomassie Brilliant Blue G-250 (Bio-Rad) at 42° C. for 6 hrsusing ³² P-labelled (kinased) synthetic DNAs. (20×SSPE is prepared bydissolving 174 g of NaCl, 27.6 g of NaH₂ PO₄ 9H2O, and 7.4 g of EDTA in800 ml of H2O. pH is adjusted to 7.4 with NaOH, volume is adjusted to 1liter, and sterilized by autoclaving). Filters are washed twice (15 min,room temperature) with 1×SSPE, 0.1% SDS. After autoradiography (Fuji RXfilm), positive colonies are located by aligning the regrown colonieswith the blue-stained colonies on the filters. DNA is sequenced by thedideoxy method, Sanger et al. Proc. Natl. Acad. Sci., Vol. 74, pg. 5463(1977). Templates for the dideoxy reactions are either single strandedDNAs of relevant regions recloned into M13mp vectors, e.g. Messing etal. Nucleic Acids Res., Vol. 9, pg. 309 (1981), or double-stranded DNAprepared by the minialkaline method and denatured with 0.2M NaOH (5 min,room temperature) and precipitated from 0.2M NaOH, 1.43M ammoniumacetate by the addition of 2 volumes of ethanol. DNA is synthesized byphosphoramidite chemistry using Applied Biosystems 380A synthesizers.Synthesis, deprotection, cleavage and purification (7M urea PAGE,elution, DEAE-cellulose chromotography) are done as described in the380A synthesizer manual.

Complementary strands of synthetic DNAs to be cloned (400 ng each) aremixed and phosphorylated with polynucleotide kinase in a reaction volumeof 50 ml. This DNA is ligated with 1 mg of vector DNA digested withappropriate restriction enzymes, and ligations are in a volume of 50 mlat room temperature for 4 to 12 hours. Conditions for phosphorylation,restriction enzyme digestions, polymerase reactions, and ligation havebeen described (Maniatis et al., cited above). Colonies are scored forlacZ+ (when desired) by plating on L agar supplemented with ampicillin,isopropyl-1-thio-beta-D-galactoside (IPTG) (0.4 mM) and5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) (40 mg/ml).

The TAC-RBS vector is constructed by filling-in with DNA polymerase thesingle BamHI site of the tacP-bearing plasmid pDR540 (Pharmacia). Thisis then ligated to unphosphorylated synthetic oligonucleotides(Pharmacia) which form a double-stranded fragment encoding a consensusribosome binding site (RBS, GTAAGGAGGTTTAAC). After ligation, themixture is phosphorylated and religated with the Sstl linker ATGAGCTCAT.This complex was then cleaved with Sstl and EcoRI, and the 173 bpfragment isolated via polyacrylamide gel electrophoresis (PAGE) andcloned into EcoRI-Sstl restricted pUC19 (Pharmacia) (as describedbelow). The sequence of the RBS-ATG-polylinker regions of the finalconstruction (called TAC-RBS) is shown in FIG. 3.

The synthetic CSIF gene is assembled into a pUC19 plasmid in eightsteps. At each step inserts free of deletions and/or inserts can bedetected after cloning by maintaining the lacZ(α) gene of pUC19 in framewith the ATG start codon inserted in step 1. Clones containing deletionand/or insertion changes can be filtered out by scoring for bluecolonies on L-ampicillin plates containing x-gal and IPTG.Alternatively, at each step sequences of inserts can be readilyconfirmed using a universal sequencing primer on small scale plasmid DNApreparations, e.g. available from Boehringer Mannheim.

In step 1 the TAC-RBS vector is digested with SstI, treated with T4 DNApolymerase (whose 3' exonuclease activity digests the 3' protrudingstrands of the SstI cuts to form blunt end fragments), and afterdeactivation of T4 DNA polymerase, treated with EcoRI to form a 173 basepair (bp) fragment containing the TAC-RBS region and having a blunt endat the ATG start codon and the EcoRI cut at the opposite end. Finally,the 173 bp TAC-RBS fragment is isolated.

In step 2 the isolated TAC-RBS fragment of step 1 is mixed withEcoRI/KpnI digested plasmid pUC19 and synthetic fragment 1A/B which, asshown below, has a blunt end at its upstream terminus and a staggeredend corresponding to an KpnI cut at its downstream terminus. This KpnIend is adjacent to and downstream of a BstEII site. The fragments areligated to form the pUC19 of step 2.

In step 3 synthetic fragment 2A/B and 3A/B (shown below) are mixed withBstEII/SmaI digested pUC19 of step 2 (after amplification andpurification) and ligated to form pUC19 of step 3. Note that thedownstream terminus of fragment 3A/B contains extra bases which form theSmaI blunt end. These extra bases are cleaved in step 4. Also fragments2A/B and 3A/B have complementary 9 residue single stranded ends whichanneal upon mixture, leaving the upstream BstEII cut of 2A/B and thedownstream blunt end of 3A/B to ligate to the pUC19.

In step 4 AflII/XbaI digested pUC19 of step 3 (after amplification andpurification) is repurified, mixed with synthetic fragment 4A/B (shownbelow), and ligated to form pUC19 of step 4.

In step 5 XbaI/SalI digested pUC19 of step 4 (after amplification andpurification) is mixed with synthetic fragment 5A/B (shown below) andligated to form the pUC19 of step 5. Note that the SalI staggered end offragment 5A/B is eliminated by digestion with HpaI in step 6.

In step 6 HpaI/PstI digested pUC19 of step 5 (after amplification andpurification) is mixed with synthetic fragment 6A/B (shown below) andligated to form the pUC19 of step 6.

In step 7 ClaI/SphI digested pUC19 of step 6 (after amplification andpurification) is mixed with synthetic fragment 7A/B (shown below) andligated to form the pUC19 of step 7.

In step 8 MluI/HindIII digested pUC19 of step 7 (after amplification andpurification) is mixed with synthetic fragments 8A/B and 9A/B andligated to form the final construction. The final construction isinserted into E. coli K-12 strain JM101, e.g. available from the ATCCunder accession number 33876, by standard techniques. After culturing,protein is extracted from the JM101 cells and dilutions of the extractsare tested for biological activity.

    AGCCCAGGCC AGGGCACCCA GTCTGAGAAC AGCTGCACCC ACTTC-                              TCGGGTCCGG TCCCGTGGGT CAGACTCTTG TCGACGTGGG TGAAG-                             - CCAGGtAACC ggtac                                                           GGTCCaTTGG c                                                                  Fragment 1A/B                                                               GtAACCTGCC TAACATGCTT CGAGATCTCC GAGATGCCTT CAGCA-                                   GACGG ATTGTACGAA GCTCTAGAGG CTCTACGGAA GTCGT-                             - GAGTGAAGACTTTCTTT                                                          CTCACTTC                                                                      Fragment 2A/B                                                                         CAAATGAAGG ATCAGCTGGA CAACTTGTTc TtAAG                                TGAAAGAAA GTTTACTTCC TAGTCGACCT GTTGAACAAg AaTTC                              Fragment 3A/B                                                               GAGTCCTTGC TGGAGGACTT TAAGGGTTAC CTGGGTTGCC AAGCC-                              CTCAGGAACG ACCTCCTGAA ATTCCCAATG GACCCAACGG TTCGG-                             - TTGTCTGAGA TGATCCAGTT TTAt                                                 AACAGACTCT ACTAGGTCAA AATaGAtC                                                Fragment 4A/B                                                               CTaGAGGAGG TGATGCCCCA AGCTGAGAAC CAAGACCCAG ACATC-                              GAtCTCCTCC ACTACGGGGT TCGACTCTTG GTTCTGGGTC TGTAG-                             - AAGGCGCATG TtAACg                                                          TTCCGCGTAC AaTTGcagct                                                         Fragment 5A/B                                                               AACTCCCTGG GGGAGAACCT GAAGACCCTC AGGCTGAGGC TACGG-                              TTGAGGGACC CCCTCTTGGA CTTCTGGGAG TCCGACTCCG ATGCC-                             - CGCTGTCATC GATctgca                                                        GCGACAGTAG CTAg                                                               Fragment 6A/B                                                               CGATTTCTTC CCTGTCAAAA CAAGAGCAAG GCCGTGGAGC AGGTG-                               TAAAGAAG  GGACAGTTTT GTTCTCGTTC CGGCACCTCG TCCAC-                             - AAGAAcGCgT gcatg                                                           TTCTTgCGcA c                                                                  Fragment 7A/B                                                               CGCGTTTAAT AATAAGCTCC AAGACAAAGG CATCTACAAA GCCAT-                                  AAATTA TTATTCGAGG TTCTGTTTCC GTAGATGTTT CGGTA-                             - GAGTGAGTTT GAC                                                             CTCA                                                                          Fragment 8A/B                                                                          ATCTTCATCA ACTACATAGA AGCCTACATG ACAAT-                               CTCAAACTG TAGAAGTAGT TGATGTATCT TCGGATGTAC TGTTA-                             - GAAGATACGA AACTGA                                                          CTTCTATGCT TTGACTtcga                                                         Fragment 9A/B                                                           

(Lower case letters indicate that a base differs from that of the nativesequence at the same site)

Example VI. Antibodies Specific for the CENKSKAVE-Peptide

50 mg of ovalbumin (OVA) and 50 mg of myoglobulin (MYO) (e.g. availablefrom Sigma) are each dissolved in 10 ml of 0.1M sodium bicarbonate, andreacted with 1 ml of 0.12 iodoacetamide solution (88 mg of iodoacetamidedissolved in 4 ml 0.1M sodium bicarbonate) for I hour at roomtemperature in a 15 ml Falcon tube (Falcon Plastics, Oxnard, Calif.), orthe like. Each reaction mixture is dialyzed overnight against 4 litersof 0.1M sodium bicarbonate at 4RC. Separately, 10 mg of CENKSKAVE isdissolved in 2 ml of 0.1M DTT (dithiotheitol) solution (containing 50 mMTris and 2.5 mM EDTA at pH8) in a 4 ml tube, incubated at 37° C.overnight; and then applied to a GF05 gel-filtration column (1.5×26.5cm) (LKB, Bromma, Sweden) and eluted with a peptide elution bufferconsisting of 0.015M acetic acid and 0.005M beta-mercaptoethanol. Threefractions of about 3.5 ml each which contained the reduced peptide areidentified by optical density at 206 nm, collected, pooled, frozen indry ice, and lyophilized overnight. Meanwhile OVA and MYO are recoveredfrom dialysis, and clarified by filtration through 0.45 micrometerfilters. OVA and MYO are activated by mixing each with 380 microlitersof N-hydroxysuccinimide ester of iodoacetic acid (NHIA) (disclosed byRector et al., in J. Immunol. Meth., Vol. 24, pg. 321 (1978)) dissolvedin tetrahydrofuran (THF) (5 mg/ml); stirring for 30 minutes at roomtemperature, and dialyzing overnight against 4 liters PBS (1.8 g NaH₂PO₄ --H₂ O, 7.2 g Na₂ HPO₄ --H₂ O; and 34 g NaCl in 4 liters H₂ O).Separately the lyophilized peptide is resuspended in 5 ml of boratereduction buffer (2 g Na₂ B₄ O₇ --10H₂ O, 17.4 g NaCl, and 336 mgEDTA-Na₂ in liter H₂ O with pH adjusted to 8.5 with concentrated HCl,deoxygenated under nitrogen for 15 minutes, after which 178 mg ascorbateis added). The dialyzed iodoacetylated OVA and MYO are recovered,separately mixed with equal volumes (preferably 2 ml) of boratereduction buffer containing the peptide, and incubated overnight at roomtemperature. The resulting conjugates are analyzed by SDS-PAGE (12.5%gel). The conjugate containing solution is diluted with PBS to 1 mg/ml,sterile filtered, and aliquotted to convenient volumes (e.g. 500microliters) for immunizations, and/or stored at 4° C. Polyclonalanti-sera against the MYO conjugate is produced in both rats and rabbits(New Zealand White). The immunization schedule for rabbits is asfollows: Initially (week 0) a 10 ml sample of serum is extracted as acontrol. One week later (week 1) 0.5 ml of peptide-carrier conjugate ismixed with 0.5 ml Freund's Complete Adjuvant and injected I.P. Threeweeks later (week 4) a booster is given consisting of 0.5 mlpeptide-carrier conjugate mixed with 0.5 ml Freund's IncompleteAdjuvant. The following week (week 5) an additional booster is given,again consisting of 0.5 ml peptide-carrier conjugate mixed with 0.5 mlFreund's Incomplete Adjuvant, followed by yet another identical boosterthe next week (week 6). On week 7, 20 ml of serum is bled from theanimal. After separating out the cellular fraction the serum assayed forpositive anti-CENKSKAVE titer by ELISA. Rat immunization proceedsimilarly except that the initial injection consists of 0.15 ml PBS and0.1 ml peptide-carrier conjugate mixed with 0.75 ml Freund's CompleteAdjuvant, boosters consisted of 0.15 ml PBS and 0.1 ml peptide-carrierconjugate mixed with 0.75 ml Freund's Incomplete Adjuvant, and only 2-3ml of serum is bled from the rat. Again, a positive anti-CENKSKAVEreaction is detected by ELISA.

The descriptions of the foregoing embodiments of the invention have beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed, and obviously many modifications and variations are possiblein light of the above teaching. The embodiments were chosen anddescribed in order to best explain the principles of the invention tothereby enable others skilled in the art to best utilize the inventionin various embodiments and with various modifications as are suited tothe particular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

Applicants have deposited separate cultures of E. coli MC1061 carryingpcD(SRα)-F115, pH5C, and pH15C, respectively, and hybridomaJES3-19F1.1.1 with the American Type Culture Collection, Rockville, Md.,USA (ATCC), under accession numbers 68027, 68191, 68192 and HB10487,respectively. These deposits were made under conditions as providedunder ATCC's agreement for Culture Deposit for Patent Purposes, whichassures that the deposits will be made available to the US Commissionerof Patents and Trademarks pursuant to 35 USC 122 and 37 CFR 1.14, andwill be made available to the public upon issue of a U.S. patent, whichrequires that the deposits be maintained. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

We claim:
 1. An isolated nucleic acid comprising from 18 to 537consecutive bases selected from the following sequence:

    ATGCACAGCT CAGCACTGCT CTGTTGCCTG GTCCTCCTGA                                      - CTGGGGTGAG GGCCAGCCCA GGCCAGGGCA CCCAGTCTGA                                 - GAACAGCTGC ACCCACTTCC CAGGCAACCT GCCTAACATG                                 - CTTCGAGATC TCCGAGATGC CTTCAGCAGA GTGAAGACTT                                 - TCTTTCAAAT GAAGGATCAG CTGGACAACT TGTTGTTAAA                                 - GGAGTCCTTG CTGGAGGACT TTAAGGGTTA CCTGGGTTGC                                 - CAAGCCTTGT CTGAGATGAT CCAGTTTTAC CTGGAGGAGG                                 - TGATGCCCCA AGCTGAGAAC CAAGACCCAG ACATCAAGGC                                 - GCATGTGAAC TCCCTGGGGG AGAACCTGAA                                     


2. The nucleic acid of claim 1 which contains from 18-60 bases.
 3. Thenucleic acid of claim 2 comprising the sequence GACTTTAAGG GTTACCTGGGTTGCCAAGCC TTGTCTGAGA TGATCCAGTT TTACCTG.
 4. The nucleic acid of claim 2comprising the sequence AGGCTACGGC GCTGTCATCG ATTTCTTCCC TGTGAAAACAAGAGCAAGGC CGTGGAGCAG.
 5. The nucleic acid of claim 1 which containsfrom 50-200 bases.
 6. An isolated nucleic acid comprising a sequenceencoding a polypeptide selected from the group consisting of:a)MLRDLRDAFS; b) VKTFFQ; c) DFKGYLGCQALSEMIQFYL; d) EVMPQAE; e)RLRRCHRFLPCENKSKAVEQ; f) CENKSKAVE; and g) EFDIFIN.
 7. The nucleic acidof claim 6 which encodes a polypeptide comprising the amino acidsequence MLRDLRDAFS.
 8. The nucleic acid of claim 7 wherein said aminoacid sequence of the polypeptide is encoded by ATGCTTCGAG ATCTCCGAGATGCCTTCAGC.
 9. The nucleic acid of claim 6 which encodes a polypeptidecomprising the amino acid sequence VKTFFQ.
 10. The nucleic acid of claim9 wherein said amino acid sequence of the polypeptide is encoded byGTGAAGACTTTCTTT.
 11. The nucleic acid of claim 6 which encodes apolypeptide comprising the amino acid sequence DFKGYLGCQALSEMIQFYL. 12.The nucleic acid of claim 11 wherein said amino acid sequence of thepolypeptide is encoded by GACTTTAAGG GTTACCTGGG TTGCCAAGCC TTGTCTGAGATGATCCAGTT TTACCTG.
 13. The nucleic acid of claim 6 which encodes apolypeptide comprising the amino acid sequence EVMPQAE.
 14. The nucleicacid of claim 13 wherein said amino acid sequence of the polypeptide isencoded by GAGGTGATGC CCCAAGCTGA G.
 15. The nucleic acid of claim 6which encodes a polypeptide comprising the amino acid sequenceRLRRCHRFLPCENKSKAVEQ.
 16. The nucleic acid of claim 15 wherein saidamino acid sequence of the polypeptide is encoded by AGGCTACGGCGCTGTCATCG ATTTCTTCCC TGTGAAAACA AGAGCAAGGC CGTGGAGCAG.
 17. The nucleicacid of claim 6 which encodes a polypeptide comprising the amino acidsequence CENKSKAVE.
 18. The nucleic acid of claim 17 wherein said aminoacid sequence of the polypeptide is encoded by TGTGAAAACA AGAGCAAGGCCGTGGAG.
 19. The nucleic acid of claim 6 which encodes a polypeptidecomprising the amino acid sequence EFDIFIN.
 20. The nucleic acid ofclaim 19 wherein said amino acid sequence of the polypeptide is encodedby GAGTTTGACA TCTTCATCAA C.